Acquired metallo-ß-lactamases and their genetic association with class 1 integrons and ISCR elements in Gram-negative bacteria.

Metallo-ß-lactamases (MBLs) can hydrolyze almost all ß-lactam antibiotics and are resistant to clinically available ß-lactamase inhibitors. Numerous types of acquired MBLs have been identified, including IMP, VIM, NDM, SPM, GIM, SIM, DIM, KHM, TMB, FIM and AIM. IMPs and VIMs are the most frequent MBLs and disseminate in members of the family Enterobacteriaceae, Pseudomonas spp. and Acinetobacter spp. Acquired MBL genes are often embedded in integrons, and some are associated with insertion sequence (IS) elements. The class 1 integrons and IS common region (ISCR) elements are usually harbored in transposons and/or plasmids, forming so-called mobile vesicles for horizontal transfer of captured genes between bacteria. Here, we review the MBL superfamily identified in Gram-negative bacteria, with an emphasis on the phylogeny of acquired MBLs and their genetic association with class 1 integrons and IS common region elements.

Epidemiology and genetics of CTX-M extended-spectrum ß-lactamases in Gram-negative bacteria.

CTX-M enzymes, the plasmid-mediated cefotaximases, constitute a rapidly growing family of extended-spectrum ß-lactamases (ESBLs) with significant clinical impact. CTX-Ms are found in at least 26 bacterial species, particularly in Escherichia coli, Klebsiella pneumoniae and Proteus mirabilis. At least 109 members in CTX-M family are identified and can be divided into seven clusters based on their phylogeny. CTX-M-15 and CTX-M-14 are the most dominant variants. Chromosome-encoded intrinsic cefotaximases in Kluyvera spp. are proposed to be the progenitors of CTX-Ms, while ISEcp1, ISCR1 and plasmid are closely associated with their mobilization and dissemination.

Up-regulation of IL-33 expression in various types of murine cells by IL-3 and IL-4.

The crucial roles of the novel cytokine IL-33 in allergic, inflammatory, infectious and autoimmune diseases are becoming characterized. However, the cytokines which regulate IL-33 expression and secretion are still largely unknown. In this study, IL-3 and IL-4 were found to up-regulate IL-33 mRNA expression in mouse peritoneal exudate cells by a two-color DNA microarray and further confirmed by real time PCR and ELISA. IL-3 and IL-4 synergistically promote IL-33 mRNA expression and IL-33 intracrine in the heterogeneous cell populations as peritoneal exudates cells, bone marrow cells and splenic cells. IL-3 and IL-4 also induced IL-33 introcrine in the peritoneal exudate cells from the macrophage-deficient op/op mice, suggesting that macrophage is not the only target of IL-3 and IL-4 in the heterogeneous peritoneal exudate cells. Furthermore, IL-3 and IL-4 were verified to promote the IL-33 intracrine in the homogeneous cell population as fibroblasts and mast cells. These results indicate that up-regulation of IL-33 expression by IL-3 and IL-4 is not a feature particular to a specific type of cells. Up to 100 cytokines were screened, but none of them stimulated the secretion or release of IL-33 in the culture system. In summary, we confirm for the first time that IL-3 and IL-4 are critical for IL-33 intracrine in murine cells of various types, indicating that IL-3 and IL-4 may play an important role in the constitutive expression of IL-33 in vivo.

Acinetobacter: a potential reservoir and dispenser for ß-lactamases.

Innate resistance and remarkable ability to acquire additional resistance determinants underline the clinical importance of Acinetobacter. Over 210 ß-lactamases belonging to 16 families have been identified in the genus, mostly in clinical isolates of A. baumannii. In this review, we update the current taxonomy of the genus Acinetobacter and summarize the ß-lactamases detected in Acinetobacter spp. with an emphasis on Acinetobacter-derived cephalosporinases (ADCs) and carbapenem-hydrolysing class D ß-lactamases (CHDLs). We also discuss the roles of integrons and insertion sequence (IS) elements in the expression and dissemination of such resistance determinants.

Identification of a plasmid-borne blaIMP-11 gene in clinical isolates of Escherichia coli and Klebsiella pneumoniae.

The acquired metallo-ß-lactamases represent a significant clinical threat due to their unrivalled hydrolysis spectrum and their resistance to therapeutic inhibitors of ß-lactamase. In this study, we identified plasmid- and integron-borne bla(IMP-11) in clinical isolates of Escherichia coli and Klebsiella pneumoniae. The bla(IMP-11) gene cassette was carried by a typical class 1 integron together with aacA1 and orfG gene cassettes. The integron, intI1-bla(IMP-11)-aacA1-orfG-qacEΔ1-sul1, was easily transferred by intraspecies and intergenus conjugation of bacteria, indicating that the integron is located on a transferable plasmid. The integrated genes were preceded by TGGACA-N(17)-TAAACT, a hybrid P(c) promoter. Similar to the wild-type donors, the transconjugants also showed reduced susceptibility or resistance to carbapenems, amikacin and kanamycin. The identical integron was detected in four bacterial strains which were genetically different but were isolated from infant inpatients in the same paediatric department. These results demonstrate the colonization of the plasmid- and integron-borne bla(IMP-11) and aacA1 in the hospital environment, highlighting the importance of surveying and controlling the spread of such resistance determinants in nosocomial pathogens.

IMP-type metallo-ß-lactamases in Gram-negative bacilli: distribution, phylogeny, and association with integrons.

Twenty-nine IMP-type ß-lactamases (IMPs) have been identified in at least 26 species of clinically important Gram-negative bacilli from more than 24 countries/regions. Most of bla(IMP) genes are harbored by class 1 integrons that are usually embedded in transposons and/or plasmids, footnoting their horizontal transfer and worldwide distribution. bla(IMP) genes usually co-exist with other resistance genes, such as aacA, catB, and bla(OXA), resulting in multi-drug resistance. Compared to other gene cassettes, 76.3% of the bla(IMP) gene cassettes are located adjacent to Pc promoter of the class 1 integrons, indicating that the bla(IMP) genes are readily expressed in most of bacterial hosts.

Epidemiology and genetics of VIM-type metallo-ß-lactamases in Gram-negative bacilli.

Metallo-ß-lactamases (MBLs) are a rapidly evolving group of ß-lactamases, which hydrolyze most ß-lactams including the carbapenems. Of the known MBLs, VIMs are one of the most common families, with 27 variants detected in at least 23 species of Gram-negative bacilli from more than 40 countries/regions. The amino acid similarities of VIM variants range from 72.9 to 99.6% with 1-72 different residues. Most of the bla (VIM)s are harbored by a class 1 integron, a genetic platform able to acquire and express gene cassettes. The integrons are usually embedded in transposons and, in turn, accommodated on plasmids, making them highly mobile. Integrons display considerable diversity, with at least 110 different structures associated with the gain and spread of the bla (VIM)s. In most instances, the bla (VIM)s co-exist with one or more other resistance genes. The processes for the identification of bacteria harboring bla (VIM)s are also discussed in this article.

Beta-lactamases identified in clinical isolates of Pseudomonas aeruginosa.

The increased prevalence of beta-lactamases in Pseudomonas aeruginosa has begun to reduce the clinical efficacy of beta-lactams against the most common opportunistic pathogen. Of over 800 beta-lactamases identified from Gram-negative bacilli, at least 120 beta-lactamases have been detected in P. aeruginosa. IMPs and VIMs are predominantly found in P. aeruginosa and like Acinetobacter spp., P. aeruginosa is also a predominant source of OXAs, indicating that P. aeruginosa is a crucial reservoir of beta-lactam resistance determinants. This review summarizes the beta-lactamases identified in clinical isolates of P. aeruginosa, with a particular focus on AmpC-type beta-lactamases, extended-spectrum beta-lactamases, and carbapenemases.

Relevance of resistance levels to carbapenems and integron-borne blaIMP-1, blaIMP-7, blaIMP-10 and blaVIM-2 in clinical isolates of Pseudomonas aeruginosa.

Molecular detection and surveillance of the resistance genes harboured by Pseudomonas aeruginosa are becoming increasingly important in assessing and controlling spread and colonization in hospitals, and in guiding the treatment of infections. This study analysed the resistance mechanisms of carbapenem-resistant clinical isolates of P. aeruginosa and identified the associated integron-borne metallo-beta-lactamase (MBL)-encoding genes. Twenty-seven imipenem (IPM)-resistant clinical isolates of P. aeruginosa were divided into three groups according to their resistance levels to carbapenems. Strains bearing bla(IMP-10) showed extremely high-level resistance to IPM, with MICs of 512-2048 microg ml(-1). By comparison, strains bearing bla(IMP-1), bla(IMP-7) and bla(VIM-2) showed an intermediate level of resistance, with MICs of 32-256 microg ml(-1). The non-MBL-producing strains showed a low level of resistance, with MICs of 8-32 microg ml(-1). The same trend in resistance levels was also observed when resistance to other carbapenems, such as meropenem and panipenem, was determined. DNA sequencing showed that the MBL-encoding gene cassettes were carried by class 1 integrons. The bla(IMP-1), bla(IMP-7) and bla(IMP-10) gene cassettes were preceded by a hybrid P(ant) promoter, TGGACA-N(17)-TAAACT, and the bla(VIM-2) gene cassette was preceded by a weak promoter, TGGACA-N(17)-TAAGCT. Most of the MBL-encoding genes were linked to one or two resistance genes encoding aminoglycoside-modifying enzymes, such as aac(6')Iae, aac(6')II, aacA7, aacC4, aadA1, aadA2 and aadA6, highlighting the multidrug-resistant properties of these clinical isolates.

Contributions of IMP-10 metallo-beta-lactamase, the outer membrane barrier and the MexAB-OprM efflux system to high-level carbapenem resistance in Pseudomonas aeruginosa.

BACKGROUND: The emergence of carbapenem-hydrolyzing metallo-beta-lactamases (MBLs) is a serious threat to clinical medication of carbapenems. We evaluated the contributions of IMP-10 MBL, the outer membrane barrier and the MexAB-OprM efflux system to high-level carbapenem resistance in Pseudomonas aeruginosa clinical isolates. METHODS: MBL-producing strains were screened by ceftazidime and mercaptoacetic acid double-disk synergy testing. Genes were determined by PCR analysis and DNA sequencing. IMP-10 MBL activity was assayed using nitrocefin as a substrate. RESULTS: The bla(IMP-10)+ strains showed high-level resistance to carbapenems, with minimum inhibitory concentrations of 512 to >4,096 microg/ml. The minimum inhibitory concentrations were decreased by the inhibitors of MBLs, sodium mercaptoacetate (SMA) and ethylenediaminetetraacetic acid (EDTA), 16 and 64 fold, respectively. However, against the activity of the prepared IMP-10 MBL, the 50% inhibitory concentrations of SMA and EDTA were 5.5 and 211.6 microM, respectively, indicating that SMA was much more effective than EDTA in blocking the enzyme activity. The contradictory results may be explained by the additional effect of EDTA as an outer membrane permeabilizer because the susceptibility of the bla(IMP)- strains to carbapenems was increased 2 fold by EDTA, but not by SMA. In the presence of carbonyl cyanide M-chlorophenylhydrazone, a proton motive force inhibitor, the susceptibility of the P. aeruginosa isolates to carbapenems was increased 1-4 fold. CONCLUSION: The acquired IMP-10 MBL is a crucial factor in the high-level resistance to carbapenems in P. aeruginosa clinical isolates, while the outer membrane barrier and the MexAB-OprM efflux system play a basic and minor role, respectively.

Potency of IMP-10 metallo-beta-lactamase in hydrolysing various antipseudomonal beta-lactams.

Limited beta-lactams show antipseudomonal activity. The rapid spread of IMP-type metallo-beta-lactamases (MBLs), which have a broad spectrum of substrates and a poor susceptibility to clinically available inhibitors, further restricts beta-lactam use. In the present study, we evaluated the potency of IMP-10 MBL in hydrolysing antipseudomonal beta-lactams currently available in the clinic. Crude IMP-10 MBL was prepared from two clinical isolates of Pseudomonas aeruginosa harbouring the bla(IMP-10) gene. The sensitivity of beta-lactams to hydrolysis by IMP-10 MBL was determined by comparing the MICs of 14 antipseudomonal beta-lactams against a susceptible strain of P. aeruginosa in the presence and absence of IMP-10 MBL. Carbapenems (imipenem, meropenem and panipenem) and extended-spectrum cephems (ceftazidime, cefoperazone, cefsulodin and cefepime) were sensitive to the hydrolysing activity of IMP-10 MBL. By comparison, the fourth-generation cephem (cefpirome), the extended-spectrum penicillins (carbenicillin, ticarcillin, piperacillin and mezlocillin) and monobactams (aztreonam and carumonam) were relatively resistant to IMP-10 MBL. The sensitivity profile of antipseudomonal beta-lactams to IMP-10 MBL generated in the present study provides a valuable reference for antibiotic selection by medical professionals.

Hyperproduction of inhibitor-susceptible TEM beta-lactamase is responsible for resistance of Serratia marcescens to beta-lactam-beta-lactamase inhibitor combinations.

BACKGROUND: Some clinical isolates of Serratia marcescens showed high-level resistance to expanded-spectrum cephalosporins alone and in combination with beta-lactamase inhibitor, but the beta-lactamase extracted from these strains was sensitive to the inhibitors. This study examines the possible mechanisms responsible for the discrepancy. METHODS AND RESULTS: Three clinical isolates of S. marcescens bore the bla(TEM) gene coded on a plasmid. This was confirmed by detection of the bla gene using PCR analysis and by transferring resistance determinants to Escherichia coli via conjugation and transformation. All of the E. coli transconjugants and transformants acquired a similar level of resistance to penicillins and narrow-spectrum cephalosporins, and showed the reduced susceptibility to expanded-spectrum cephalosporins alone and in combination with clavulanate. As a result of the highly constitutive expression of the TEM gene, up to 247-690 U of beta-lactamase were produced by 10(10) cells of wild-type S. marcescens and the transconjugants and transformants. In the presence of 0.24 U/ml of TEM enzyme, the minimum inhibition concentrations of cefotaxime against E. coli ATCC 25922 increased from 0.125 to 512 microg/ml. The TEM-type beta-lactamase extracted from these strains was sensitive to clavulanate, and 62.2-92.1% of its activity was inhibited after preincubation with 0.1 mM clavulanate. CONCLUSION: The TEM-type beta-lactamase plays a critical role in the resistance of S. marcescens to beta-lactams, and the hyperproduction of inhibitor-susceptible TEM beta-lactamase is responsible for the resistance to beta-lactam-beta-lactamase inhibitor combinations.

Regulation of mast cell development by inflammatory factors.

Mast cells are potent effectors playing a key role in IgE-associated hypersensitivity reactions, allergic disorders, inflammation and protective immune responses. Mast cell development in vivo occurs mainly in non-hematopoietic microenvironments and increased mast cell numbers can be seen in various inflammatory diseases and pathologic conditions. SCF (also known as kit ligand or KitL) and c-kit signaling are essential for both human and murine mast cell development, while IL-3 is required for murine mast cell hyperplasia that occurs in response to various stimuli. Besides SCF and IL-3, the cytokines IL-4, IL-9, IL-10 and IL-13 are also called mast cell growth factors due to their actions synergistically promoting mast cell proliferation and differentiation in the presence of SCF or IL-3. These cytokines alone however are unable to support neither the proliferation nor survival of mast cells. Most research has focused on examining the direct effects of the above cytokines on mast cells or their precursors. However, it is difficult to explain the process of mast cell development only in terms of the above mast cell growth factors. A series of experiments in our laboratory and by others has revealed that inflammatory mediators and cytokines, as triggers or regulators, are also crucial for mast cell development. This review summarizes recent progress in our understanding of how various inflammatory factors regulate mast cell development, with particular focus on the effects of prostaglandin E (PGE), TNF-alpha, IL-6, IFN-gamma and an unknown apoptosis-inducing factor produced by IL-4-stimulated macrophages.

Characterization of imipenem-resistant Serratia marcescens producing IMP-type and TEM-type beta-lactamases encoded on a single plasmid.

To evaluate the roles of blaIMP and blaTEM genes in the resistance of Serratia marcescens against beta-lactams and to find the spreading ways of these genes, 19 clinical isolates of imipenem-resistant Serratia marcescens were analyzed. Six strains bore blaIMP and blaTEM genes on a single plasmid, as confirmed by transferring resistance determinants via conjugation and transformation, and by detecting bla genes with PCR analysis. The six strains showed two different genomic patterns on pulsed-field gel electrophoresis. All the transconjugants and transformants gained high-level resistance to ampicillin, cephalexin, cefoxitin and cefotaxime, and showed a reduced susceptibility to imipenem, but maintained full susceptibility to aztreonam. In addition, the expressions of blaIMP and blaTEM genes were constitutive, either in Serratia marcescens clinical isolates or in their transconjugants and transformants. These findings may explain the rapid spread of the above resistance determinants among Enterobacteriaceae via transmissible plasmids in the clinical setting.

Interleukin-4-triggered, STAT6-dependent production of a factor that induces mouse mast cell apoptosis.

IL-4 can suppress mast cell development from mouse spleen, bone marrow and peritoneal cells by an indirect process that is dependent on the presence of macrophages. Mast cells undergo apoptosis when exposed to supernatants collected from cultures of IL-4-stimulated peritoneal cells due to the IL-4-induced production of an apoptosis-inducing factor in the cultures. This effect of IL-4 is shown to be dependent on STAT6 signaling, because IL-4 and IL-13 do not suppress mast cell development from the spleen and peritoneal cells of STAT6-/- mice. Moreover, supernatants from cultures of IL-4- and IL-13-stimulated peritoneal cells of STAT6-/- mice do not exhibit apoptosis-inducing activity. We confirm, by using deficient mice, neutralizing antibodies and recombinant cytokines, that IL-4-induced apoptosis is not related to the well-known apoptosis-inducing factors Fas, Fas ligand, TNF-alpha, TRAIL, TGF-beta or perforin. These results demonstrate a novel mechanism whereby IL-4 and IL-13 can suppress mast cell development by inducing the production of an apoptosis-inducing factor from macrophages.